3.4 kb mitochondrial dna deletion for use in the detection of cancer

ABSTRACT

The present invention broadly claims a method for detecting cancer in an individual. The method comprises detecting a deletion in the nucleic acid sequence between residues 10743 and 12125 in mitochondrial DNA. The method comprises obtaining a biological sample from the individual; extracting the mitochondrial DNA (mtDNA) from the sample; quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence between residues 10743 and 14125 of the mtDNA genome; and comparing the amount of mtDNA in the sample having the deletion to at least one known reference sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §365 (c) as acontinuation of U.S. patent application Ser. No. 12/748,120 filed onMar. 26, 2010 which application claims the benefit under 35 U.S.C. §120as a continuation-in-part of U.S. patent application Ser. No. 11/975,390filed Oct. 18, 2007 which is a continuation of PCT/CA2006/000652, filedApr. 18, 2006 which application claims the benefit under 35 U.S.C. §119(e) from U.S. Provisional Patent Application Nos. 60/672,016 filed Apr.18, 2005, 60/721,522 filed Sep. 29, 2005, and 60/789,872 filed Apr. 7,2006. In addition, this application claims priority under 35 U.S.C. §365(c) as a continuation of PCT application PCT/CA2007/001711, filed Sep.26, 2007, published in English. The entire disclosures of theseapplications are incorporated by reference herein.

REFERENCE TO SEQUENCE IDENTIFICATION LISTING

The present application includes a sequence identification listing in.txt format as follows:

Filename: Sequence Listing re PCT International Patent Appl. No.

-   -   PCT_CA2007_(—)001711.txt

Size: 26.8 KB Date Created: Mar. 22, 2010

This sequence identification listing is hereby expressly incorporated byreference in its entirety in the present application.

FIELD OF THE INVENTION

This invention is related to the field of mitochondrial genomics. Inparticular it is related to a 3.4 kb deletion in the mitochondrialgenome and its utility as an indicator of cancer.

BACKGROUND OF THE INVENTION

Mitochondrial DNA (MtDNA) as a Diagnostic Tool

MtDNA sequence dynamics are important diagnostic tools. Mutations inmtDNA are often preliminary indicators of developing disease, oftenassociated with nuclear mutations, and act as biomarkers specificallyrelated to: disease, such as but not limited to, tissue damage andcancer from smoking and exposure to second hand tobacco smoke (Lee etal., 1998; Wei, 1998); longevity, based on accumulation of mitochondrialgenome mutations beginning around 20 years of age and increasingthereafter (von Wurmb, 1998); metastatic disease caused by mutation orexposure to carcinogens, mutagens, ultraviolet radiation (Birch-Machin,2000); osteoarthritis; cardiovascular, Alzheimer, Parkinson disease(Shoffner et al., 1993; Sherratt et al., 1997; Zhang et al, 1998); ageassociated hearing loss (Seidman et al., 1997); optic nerve degenerationand cardiac dysrhythmia (Brown et al., 1997; Wallace et al., 1988);chronic progressive external exophthalmoplegia (Taniike et al., 1992);atherosclerosis (Bogliolo et al., 1999); papillary thyroid carcinomasand thyroid tumours (Yeh et al., 2000); as well as others (e.g. Naviaux,1997; Chinnery and Turnbull, 1999).

Mutations at specific sites of the mitochondrial genome can beassociated with certain diseases. For example, mutations at positions4216, 4217 and 4917 are associated with Leber's Hereditary OpticNeuropathy (LHON) (Mitochondrial Research Society; Huoponen (2001);MitoMap). A mutation at 15452 was found in 5/5 patients to be associatedwith ubiquinol cytochrome c reductase (complex III) deficiency (Valnotet al. 1999).

Specifically, these mutations or alterations include point mutations(transitions, transversions), deletions (one base to thousands ofbases), inversions, duplications, (one base to thousands of bases),recombinations and insertions (one base to thousands of bases). Inaddition, specific base pair alterations, deletions, or combinationsthereof have been found to be associated with early onset of prostate,skin, and lung cancer, as well as aging (e.g. Polyak et al., 1998),premature aging, exposure to carcinogens (Lee et al., 1998), etc.

Prostate Cancer

Prostate cancer is a frequently diagnosed solid tumour that most likelyoriginates in the prostate epithelium (Huang et al. 1999). In 1997,nearly 10 million American men were screened for prostate specificantigen (PSA), the presence of which suggests prostate cancer (Woodwell,1999). Indeed, this indicates an even higher number of men screened byan initial digital rectal exam (DRE). In the same year, 31 million menhad a DRE (Woodwell, 1999). Moreover, the annual number of newlydiagnosed cases of prostate cancer in the United States is estimated at179,000 (Landis et al., 1999). It is the second most commonly diagnosedcancer and second leading cause of cancer mortality in Canadian men. In1997 prostate cancer accounted for 19,800 of newly diagnosed cancers inCanadian men (28%) (National Cancer Institute of Canada). It isestimated that 30% to 40% of all men over the age of forty-nine (49)have some cancerous prostate cells, yet only 20% to 25% of these menhave a clinically significant form of prostate cancer (SpringNet—CEConnection, internet, www.springnet.com/ce/j803a.htm). Prostate cancerexhibits a wide variety of histological behaviour involving bothendogenous and exogenous factors, i.e. socio-economic situations, diet,geography, hormonal imbalance, family history and genetic constitution(Konishi et al. 1997; Hayward et al. 1998). Although certain mtDNAalterations have been previously associated with prostate cancer, theneed exists for further markers for the detection of prostate cancer.

3.4 Kb mtDNA Deletion and the Detection of Prostate Cancer.

In the applicant's pending PCT application bearing publication no.WO/06/111029 (the entire contents of which are incorporated herein byreference) a deletion of a 3379 bp segment of mtDNA was identifiedthrough full mitochondrial genome amplification of prostate tissue. The3379 bp deletion (referred to as the 3.4 kb deletion) was determined tobe located between nucleotides 10744-14124 of the mitochondrial genome.It was determined that the detection of this deletion could be used inthe diagnosis of prostrate cancer when tissue samples are tested.

The 3.4 kb deletion removes all or part of the following genes from themtDNA genome: (i) NADH dehydrogenase subunit 4L, (ii) NADH dehydrogenasesubunit 4, (iii) NADH dehydrogenase subunit 5, (iv) tRNA histidine, (v)tRNA serine2, and (vi) tRNA leucine2.

Breast Cancer

Breast cancer is a cancer of the glandular breast tissue and is thefifth most common cause of cancer death. In 2005, breast cancer caused502,000 deaths (7% of cancer deaths; almost 1% of all deaths) worldwide(World Health Organization Cancer Fact Sheet No. 297). Among womenworldwide, breast cancer is the most common cancer and the most commoncause of cancer death (World Health Organization Cancer Fact Sheet No.297). Although certain mtDNA alterations have been previously associatedwith breast cancer, for example in Parrella et al. (Cancer Research: 61,2001), the need exists for further markers for the detection of breastcancer.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method of detectinga cancer in an individual comprising;

a) obtaining a biological sample from the individual;

b) extracting mitochondrial DNA, mtDNA, from the sample;

c) quantifying the amount of mtDNA in the sample having a deletion inthe nucleic acid sequence spanning approximately residues 10744 and14124 of the mtDNA genome;

d) comparing the amount of mtDNA in the sample having the deletion to atleast one known reference value.

In one embodiment, the present invention provides a method of detectinga cancer in an individual comprising;

a) obtaining a biological sample from the individual;

b) extracting mitochondrial DNA, mtDNA, from the sample;

c) quantifying the amount of mtDNA in the sample having a deletion inthe nucleic acid sequence spanning approximately residues 10744 and14124 of the mtDNA genome;

d) comparing the amount of mtDNA in the sample having the deletion tothe amount of the deletion in a reference sample of mtDNA from knownnon-cancerous tissue or body fluid;

wherein an elevated amount of the deletion in the biological samplecompared to the reference sample is indicative of cancer.

In one embodiment, the present invention provides a method of detectinga cancer in an individual comprising;

a) obtaining a biological sample from the individual;

b) extracting mitochondrial DNA, mtDNA, from the sample;

c) quantifying the amount of mtDNA in the sample having a deletion inthe nucleic acid sequence spanning approximately residues 10744 and14124 of the mtDNA genome;

d) comparing the amount of mtDNA in the sample having the deletion tothe amount of the deletion in a reference sample of mtDNA from knowncancerous tissue or body fluid;

wherein a similar level of the deletion in the biological samplecompared to the reference sample is indicative of cancer.

In one embodiment, the present invention provides a method of monitoringan individual for the development of a cancer comprising;

a) obtaining a biological sample;

b) extracting mtDNA from the sample;

c) quantifying the amount of mtDNA in the sample having a deletion inthe nucleic acid sequence spanning approximately residues 10744 and14124 of the mtDNA genome;

d) repeating steps a) to c) over a duration of time;

e) wherein an increasing level of the deletion over the duration of timeis indicative of cancer.

In one embodiment, the present invention provides a method of detectinga cancer in an individual comprising;

a) obtaining a biological sample from the individual;

b) extracting mitochondrial DNA, mtDNA, from the sample;

c) quantifying the amount of mtDNA in the sample having a sequencecorresponding to the sequence identified in SEQ ID NO: 1;

d) comparing the amount of mtDNA in the sample corresponding to SEQ IDNO: 1 to at least one known reference value.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

An embodiment of the invention will now be described by way of exampleonly with reference to the appended drawings wherein:

FIG. 1 is a schematic diagram showing the design and sequence of aprimer useful for the detection of the 3.4 kb deletion. The primer (SEQID NO: 2) binds to bases 10729-10743/14125-14139 of the mtDNA genome,wherein the portion of the primer that binds to bases 10729-10743 isdepicted in gray in the upper left corner of the figure (nucleotides1-15 of SEQ ID NO: 2) and the portion of the primer that binds to bases14125-14139 is depicted in white in the lower right corner of the figure(nucleotides 16-30 of SEQ ID NO: 2).

FIG. 2 is a graph showing a comparison of cycle threshold betweenmalignant and symptomatic benign participants in the 3.4 kb study.

FIG. 3 is a graph showing cycle threshold as related to Example 1.

FIG. 4 shows a ROC curve illustrating the specificity and sensitivity ofone embodiment of the present invention.

FIG. 5 shows a ROC curve illustrating the specificity and sensitivity ofanother embodiment of the present invention.

FIG. 6 shows real-time PCR data relating to 3.4 kb mtDNA deletion levelsassociated with breast cancer.

FIG. 7 shows a ROC curve illustrating the specificity and sensitivity ofanother embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “cycle threshold” (C_(T)) is the point at which targetamplification using real-time PCR rises above background, as indicatedby a signal such as a fluorescence signal. The C_(T) is inverselyrelated to the quantity of the sequence being investigated.

As defined herein, “sensitivity” refers to the fraction of truepositives (true positive rate) results obtained using the method of thepresent invention.

As defined herein, “specificity” refers to the fraction of falsepositives (false positive rate) results obtained using the method of thepresent invention.

In one embodiment of the present invention, methods are provided formonitoring and diagnosing cancer through the detection andquantification of the aforementioned 3.4 kb mtDNA deletion. For example,the present invention may be used for detecting the presence ofpre-neoplasia, neoplasia and progression towards potential malignancy ofprostate cancer and breast cancer. In one aspect, the present inventioninvolves the detection and quantification of the 3.4 kb mtDNA deletion(SEQ ID NO:1) for the detection, diagnosis, and/or monitoring of cancer.In this method, mtDNA is extracted from a biological sample (for examplebody tissue, or body fluids such as urine, prostate massage fluid). Theextracted mtDNA is then tested in order to determine the levels (ie.quantity) of the 3.4 kb deletion in the sample. In tests conducted bythe present inventors, the levels of the deletion were found to beelevated in samples obtained from subjects with cancer when compared tosamples obtained from subjects without cancer. Based on the informationand data supplied below, the inventors have concluded that elevatedlevels of the 3.4 kb deletion in the mtDNA is indicative of cancer.

As disclosed in PCT WO/06/111029, the 3.4 kb deletion spansapproximately nucleotides 10744 to 14124 of the mtDNA genome. The mtDNAgenome is listed as SEQ ID NO:8 (Genbank accession no. AC 000021). Theinventors have determined, as provided by example below, that thisdeletion is also associated with cancer and in particular prostate andbreast cancer. Therefore, such deletion provides an accurate biomarkerand, therefore, a valuable tool for the detection, diagnosis, ormonitoring of cancer in at least these tissues.

The deletion results in the creation of two deletion monomers, one of3.4 kb in size (small sublimon) and one of approximately 12.6 kb in size(large sublimon). The occurrence of the deletion may be detected byeither identifying the presence of the small sublimon, or by determiningthat the 3.4 kb sequence has been deleted from the large sublimon.

As discussed above, the deletion is approximately 3379 bp, and comprisesgenes encoding NADH dehydrogenase subunit 4L, NADH dehydrogenase subunit4, NADH dehydrogenase subunit 5, tRNA histidine, tRNAserine2, and tRNAleucine2.

In one embodiment, samples of, for example prostate tissue, prostatemassage fluid, urine or breast tissue, are obtained from an individualand tested over a period of time (eg. years) in order to monitor thegenesis or progression of cancer. Increasing levels of the 3.4 kbdeletion over time could be indicative of the beginning or progressionof cancer.

Age related accumulation of the 3.4 kb mtDNA deletion may predispose anindividual to, for example, prostate cancer or breast cancer, which isprevalent in middle aged and older men, and middle aged and older women,respectively. According to one aspect of the invention, a method isprovided wherein regular cancer screening may take place by monitoringover time the amount of the 3.4 kb deletion in body tissues such asbreast tissue or body fluids such as prostate massage fluid, or urine.

The system and method of the present invention may be used to detectcancer at an early stage, and before any histological abnormalities. Forexample, the system and method of the present invention may be used todetect pre-neoplasia in breast tissue.

The following primer sequences are preferred for the detection of the3.4 kb deletion:

3.4 forward (binds to bases 10729-10743/14125-14139 of the mtDNA genome) (SEQ ID NO: 2)5′-TAGACTACGTACATACTAACCCTACTCCTA-3′;3.4 reverse (binds to bases 14361-14379 of the   mtDNA genome)(SEQ ID NO: 3) 5′-GAGGTAGGATTGGTGCTGT-3′. 

In one embodiment of the present invention, a pair of amplificationprimers are used to amplify a target region indicative of the presenceof the 3.4 kb deletion. In this embodiment, one of the pair ofamplification primers overlaps a spliced region of mtDNA after deletionof the 3.4 kb sequence has occurred (ie. a splice at a position spanningapproximately residues 10744 and 14124 of the mtDNA genome). Therefore,extension of the overlapping primer can only occur if the 3.4 kb sectionis deleted.

In another embodiment of the present invention, a pair of amplificationprimers are used to amplify a target region associated with the deleted3.4 kb sequence. The deleted 3.4 kb sequence, upon deletion, may reformas a circular mtDNA molecule. In this embodiment, one of the pair ofamplification primers overlaps the rejoining site of the ends of the 3.4kb sequence. Thus, an increase in the amount of the 3.4 kb moleculedetected in a sample is indicative of cancer. The below primer pair ispreferred for the detection of the deleted 3.4 kb nucleic acid.

(SEQ ID NO: 9) Forward 14115/10755 5′-CCCACTCATCACCTAAACCTAC-3′(SEQ ID NO: 10) Reverse 10980R 5′-GGTAGGAGTCAGGTAGTTAG-3′.

In one aspect of the invention, a kit for diagnosing cancer, for exampleprostate or breast cancer, comprising means for extraction of mtDNA,primers having the nucleic acid sequences recited in SEQ ID NOS: 2 and3, or SEQ ID NOS: 9 and 10, reagents and instructions, is provided.

Another aspect of the invention provides methods for confirming orrefuting the presence of a cancer biopsy test from a biopsy sample (eg.prostate or breast cancer), comprising: obtaining non-cancerous tissuefrom a biopsy sample; and detecting and quantifying the amount of the3.4 kb mtDNA deletion in the non-diseased tissue.

In one embodiment the present invention provides a method for screeningindividuals for prostate or breast cancer from a body fluid samplecomprising; obtaining a body fluid sample, and detecting and quantifyingthe level of the 3.4 kb mtDNA deletion in the body fluid.

Although real-time quantitative PCR methods, as described in theexamples below, represent the preferred means for detecting andquantifying the presence or absence of the 3.4 kb deletion, othermethods that would be well known to an individual of skill in the artcould also be utilized. For example quantification of the deletion couldbe made using Bio-Rad's Bioplex™ System and Suspension Array technology.Generally, the method requires amplification and quantification ofsequences using any known methods.

The examples provided below illustrate that not only can this deletionbe used for the detection of prostate cancer in prostate tissue, but canalso be used to detect the presence of cancer in other biologicalsamples, for example prostate massage fluid, urine, and breast tissue.Based on the findings in these examples, the 3.4 kb mtDNA deletion maybe used as a biomarker for cancer.

The various examples provided illustrate a difference in the amount ofmtDNA having the 3.4 kb deletion between samples obtained from subjectshaving cancer, and subjects without cancer. The amount of the 3.4 kbdeletion was found to be higher in the samples obtained from subjectshaving cancer. This determination was made by comparing the amount ofthe 3.4 kb deletion in the test samples with amounts from known cancercells and/or known non-cancer cells.

Example 1 3.4 Kb Deletion in the mtDNA of Prostate Tissue

A deletion of approximately 3.4 kilobases (kb) was identified throughfull mitochondrial genome amplification of fresh frozen prostate tissue.Using linear regression, the size of the deletion was estimated to bebetween 3000 base pairs (bp) and 3500 bp. Two possible candidatedeletions were identified using Mitomap™ (Brandon, M. C., Lott, M. T.,Nguyen, K. C., Spolim, S., Navathe, S. B., Baldi, P. & Wallace, D. C.,MITOMAP: a human mitochondrial genome database—2004 update. NucleicAcids Research 33 (Database Issue):D611-613, 2005; www.mitomap.org), the3397 bp deletion at 9574-12972, and the 3379 bp deletion at 10744-14124.In order to determine which of the two deletions was associated withprostate cancer, if either, a forward primer which bridged the deletionjunction was developed for each of the two candidates, ensuring that theprimer extended further than the repeat regions that flank thedeletions. FIG. 1 is a schematic diagram showing the design and sequenceof the primer (ie. SEQ ID NO: 2). Positive amplification results for theamplicon corresponding to the 3379 bp deletion (referred to as the 3.4kb deletion) at 10744-14124 were obtained.

As indicated above, the 3.4 kb deletion removes all or part of thefollowing genes: (i) NADH dehydrogenase subunit 4L, (ii) NADHdehydrogenase subunit 4, (iii) NADH dehydrogenase subunit 5, (iv) tRNAhistidine, (v) tRNA serine2, and (vi) tRNA leucine2.

The 3.4 kb deletion was determined to be present in 91% of 33 freshfrozen prostate samples. With the specific deletion primers, formalinfixed tissues were tested in order increase the n value.

The present investigators sequenced entire mitochondrial genomes from 32tissue samples microdissected by laser capture microdisection and 12needle biopsies from histologically normal prostates. Archived tissuesections from each of these samples were used for the following study.1-2 serial sections were removed from each sample. DNA was extractedfrom each sample in its entirety rather than as a microdissection. Thus,each sample consisted of a mixture of glandular prostate tissue as wellas stromal prostate tissue. This extraction was performed using Qiagen'sQIAamp™ DNA Mini Kit (Cat #51304). Following extraction the samples werequantified using a Nano-Drop™ spectrophotometer and the concentrationswere subsequently normalized to 2 ng/ul. Each sample was amplified using20 ng input DNA and an iQ™ SYBR Green Supermix™ kit (Bio-RadLaboratories Inc.) Reactions were run on an Opticon® 2 two colourreal-time PCR system (MJ Research).

As shown in FIG. 2, a distinct difference was observed in cyclethreshold and, by extension, quantity of the deletion between themalignant prostate samples and the symptomatic benign prostate samples.Malignant samples exhibited a consistently earlier cycle threshold thanthe benign samples.

Example 2 3.4 Kb Deletion Blinded Study—Comparison of Cycle Threshold

An additional 21 prostate tissue samples were selected, 10 of which werebenign and 11 of which were malignant. The pathological status wasdetermined by needle biopsies conducted by a qualified pathologist. Thesamples were blinded such that the present investigators were unaware oftheir pathological status when they conducted this test. The presentinvestigators were able to predict pathological status correctly in 81%of the cases by examining the cycle threshold. Of the 4 incorrect calls,two were malignant samples that were determined to be benign and 2 werebenign samples that were determined to be malignant. Follow-up clinicalinformation for the 2 individuals in the latter scenario was requestedfrom the physician to determine if they had been diagnosed with prostatecancer subsequent to the needle biopsy results used for this study. Oneof the individuals who originally produced a benign sample but waspredicted by this study to have a malignancy subsequently produced amalignant sample. As a result, one of the false positives became a truepositive. Therefore, pathological status was predicted correctly in 86%of the cases examined in this study. The ultimate positive predictivevalue (PPV, where PPV=true positives/(true positives+false positives))for this study was 91% and the negative predictive value (NPV, whereNPV=true negatives/(true negatives+false negatives)) was 80%.

Example 3 3.4 Kb Deletion Study—Methods (n=76)

Seventy-six prostate tissue samples were examined for the 3.4 kbdeletion in this study. All tissue samples were formalin-fixed, 25 beingmalignant, 12 being normal, and 39 having benign prostatic disease asshown histologically. Of the latter group more then half hadhyperplasia. All specimens were needle biopsies taken from theinvestigators' tissue archives.

Prostate Specimens

A tapelift was performed on each slide using Prep-Strips (CatalogueNumber LCMO207) from Arcturus Bioscience Inc. This allowed the removalof any particulate matter or non-adhering tissue from the slide prior toDNA extraction. With the tissue still on the slides, the slides wererinsed with PBS (Phosphate Buffered Saline Solution) to remove as muchfixative as possible. The 1-2 needle biopsy sections on the slides werescraped into sterile microcentrifuge tubes using individually wrapped,sterilized surgical razor blades. DNA was then isolated and purifiedusing a QIAamp® DNA Mini Kit (Qiagen, Cat. #51304) according tomanufacturer's specifications. A negative extract control was processedin parallel with the slide extractions as a quality control checkpoint.The total concentration of DNA and purity ratio for each sample wasdetermined by spectrophotometry (Nano-Drop™ ND-1000) and dilutions of 2ng/μl were prepared for the purpose of Quantitative Polymerase ChainReaction (qPCR).

Primers (Oligonucleotides)

Purified oligonucleotide primers were chemically synthesized byInvitrogen (California, USA). The sequences of the primers and theexpected sizes of the PCR products amplified are listed in Table 1. Inaddition, PCR analysis for mtDNA deletions included positive controls(DNA from a source known to carry the mutant mtDNA). Each primer setwith the exception of TNF (tumor necrosis factor) were checked against amitochondria-free rho 0 cell line to confirm the absence of pseudogenecoamplification.

TABLE 1 Amplification Primers. Length of amplified  Position productPrimer Amplified  (base Pair 5′-3′ pairs) 3.4 Deletion Real- 10729-14379273 Time (less 3379 bp  at 10744-14124) 12s mtDNA 708-945 238 TNF3756-3886 131 3.4 forward (10729-10743-14125-14139)5′TAGACTACGTACATACTAACCCTACTCCTA-3′ SEQ ID NO: 2 3.4 reverse(14361-14379) 5′-GAGGTAGGATTGGTGCTGT-3′ SEQ ID NO: 3 12s forward(708-728) 5′-CGTTCCAGTGAGTTCA CCCTC-3′ SEQ ID NO: 4 12s reverse(923-945) 5′-CACTCTTTACGCCGGCTTCTATT-3′ SEQ ID NO: 5 TNF forward(3756-3775) 5′-CCTGCCCCAATCCCTTTATT-3′ SEQ ID NO: 6 TNF reverse(3866-3886) 5′-GGTTTCGAAGTGGTGGTC TTG-3′ SEQ ID NO: 7

Real-Time Polymerase Chain Reaction

Three separate PCRs were performed on each sample. Each reaction was 25μl total volume and included template DNA, one pair of primers (12s or3.4 Deletion or TNF), an iQ™ SYBR Green Supermix™ kit (Catalogue Number170-8882, Bio-Rad Laboratories Inc.) and distilled deionized water(ddH₂O). The TNF (tumor necrosis factor) comprised single copy nucleargene primers, and 12s comprised total mitochondrial genome primers. Thevolume and concentrations for template DNA, primers, and reaction bufferare listed below.

TABLE 2 qPCR Components. Concentration per Volume per Reagent ReactionReaction Reaction Buffer 1X 12.5 μl Primer (forward 250 nM 0.0625 μl ofeach and reverse) 100 umole stock ddH₂O N/A 2.375. μl Template DNA 20 ng10.0 μl Total 25 μl

The cycling parameters for each amplicon are listed in Table 3.

TABLE 3 Cycling Parameters. Step Temperature (° C.) Duration 1 95 3 min2 95 30 sec 3 66 (3.4 deletion primers) or 30 sec 61.5 (12s primers) or61.5 (TNF primers) 4 72 30 sec 5 Plate Read 6 72 10 min 7 Melting Curve50° C.-110° C. reading every 1° C. 3 sec Repeat steps 2-5, 44 times fora total of 45 cycles.

Thermal cycling, real-time detection and analysis of the reactions wascarried out using a DNA Engine Opticon® 2 Continuous FluorescenceDetection System equipped with Intuitive Opticon Monitor™ software (MJResearch Inc.). The standard curve method was utilized for DNAquantification. A set of serial dilutions (10⁶, 10⁵, 10⁴, 10³, 10², 10¹)of three purified PCR generated templates, one product for the 3.4deletion, one for the 12s primers, and one for TNF. From this, threedifferent standard curves were generated showing the number of copies oftotal mtDNA (12s amplicon-total mitochondrial genome primers), theamount of mtDNA having the 3.4 kb deletion, or total nuclear DNA(TNF-single copy nuclear gene primers). The C_(T) values of the sampleswere then converted to the number of DNA copies by comparing the sampleC_(T) to that of the standards. The 3.4 deletion was considered to beabsent or at low levels if the deletion was not detected within 37cycles.

The determination of malignancy is based upon the quantity of the 3.4 kbdeletion present in the normalized sample as indicated by the locationof the cycle threshold. This location may be either absolute, as ingreater than 25 cycles but less than 35 cycles, or more likely a ratiobetween the total mitochondrial DNA present as indicated by the 12samplicon, and the 3.4 kb deletion. This may be expressed as a per centof the total mitochondrial DNA. The number of cells, as represented bythe TNF amplicon, may be incorporated to refine the distinction betweenbenign and malignant tissues.

In order to automate the analyses of these samples, bioinformatics toolswere employed. The three variables that were considered for theseanalyses were the cycle threshold C_(T) of Tumour Necrosis Factor (TNF),total pecies of mitochondria that contain those specific primer sites,and those mitochondria that harbour the deletion of interest.

Cluster Analysis

The clustering was not normalized nor were logarithmic functions useddue to the similar and small range of data.

FIG. 3 shows the actual movement and trends of the data. The x-axis isthe patient number and the y-axis is the cycle threshold obtained fromreal time PCR.

It is important to note that the higher the cycle threshold is, thelower amount of the deletion is present.

The general trend shown in FIG. 3 is based upon the differences/ratiosbetween the variables of Deletion, Total, and TNF. The deletion is lowto absent for the benign/normal samples (right side) and increases(toward the left) with abnormal benign and malignant samples. Theabnormal benign and malignant samples begin to differentiate themselvesfrom each other based on the cycle threshold ratio of Deletion to TNF.

Supervised Learning

Supervised learning is based on the system trying to predict outcomesfor known samples. Half of the data was used to train and the other halfto test the algorithm. Supervised learning compares its predictions tothe target answer and “learns” from its mistakes. But, if the predictedoutput is higher or lower than the actual outcome in the data, the erroris propagated back through the system and the weights are adjustedaccordingly.

Data SET: 5% to 35%—Benign

-   -   35% to 65%—Hyperplasia    -   65% to 95%—Malignant

Artificial Neural Network (ANN) Algorithm (shown schematically below):

-   -   Half of Data set used for Training ANN    -   Other half used to compare the accuracyAccuracy=Compare expected        data set with obtained data set→86.6%

Supervised Learning of Deletion Data using Artificial Neural Network(ANN)

Three Classifications:

-   -   Benign    -   Hyperplasia    -   Malignant

Three variables for each classification were used based on Real Time PCRCycle Threshold C_(T):

Tumour Necrosis Factor (TNF)—Nuclear copy control.

Total Mitochondria—Mitochondria copy control

Deletion—Mitochondria in the deleted state.

Results:

Half of data set is used to train the ANN, and the remaining half isused to compare the accuracy.

Three Classification Accuracy=86.6%

Positive Predictive Value (PPV);

Benign to Malignant=88.2%

Negative Predictive Value (NPV)

Benign to Malignant=76.5%

Example 4 3.4 Kb Deletion in mtDNA Associated with Breast Cancer

18 samples were tested from malignant and benign breast tissue, 9 beingmalignant and 9 being benign, for the presence of the aforementioned 3.4kb deletion. Samples were classified as either malignant or benign usingconventional histopathological analysis.

DNA was isolated and purified from the samples using a QIAamp DNA MiniKit (Qiagen, Cat. #51304) according to manufacturer's specifications.

Purified oligonucleotide primers were chemically synthesized byInvitrogen (California, USA). The sequences of the primers and theexpected sizes of the PCR products amplified are listed in Table 1above.

Real-Time Polymerase Chain Reaction

Three separate PCRs were performed on each sample. Each reaction was 25μl total volume and included template DNA, one pair of primers (12s or3.4 Deletion or TNF), an iQ™ SYBR Green Supermix kit (Catalogue Number170-8882, Bio-Rad Laboratories Inc.) and distilled deionized water(ddH₂O). The TNF (tumor necrosis factor) comprised single copy nucleargene primers, and 12s comprised total mitochondrial genome primers. Thevolume and concentrations for template DNA, primers, and reaction bufferare listed below:

TABLE 4 qPCR Components. Concentration per Volume per Reagent ReactionReaction Reaction Buffer 1X 12.5 μl Primer (forward 250 nM 0.0625 μl ofeach and reverse) 100 μmole stock ddH₂O N/A 2.375. μl Template DNA 20 ng10.0 μl Total 25 μl

The cycling parameters for each amplicon are listed in Table 5.

TABLE 5 Cycling Parameters. Step Temperature (° C.) Duration 1 95 3 min2 95 30 sec 3 66 (3.4 deletion primers) or 30 sec 61.5 (12s primers) or61.5 (TNF primers) 4 72 30 sec 5 Plate Read 6 72 10 min 7 Melting Curve50° C.-110° C. reading every 1° C. 3 sec Repeat steps 2-5, 44 times fora total of 45 cycles.

Thermal cycling, real-time detection and analysis of the reactions wascarried out using a DNA Engine Opticon® 2 Continuous FluorescenceDetection System equipped with Intuitive Opticon Monitor™ software (MJResearch Inc.). The standard curve method was utilized for DNAquantification. A set of serial dilutions (10⁶, 10⁵, 10⁴, 10³, 10², 10¹)of three purified PCR generated templates were performed, one productfor the 3.4 deletion, one for the 12s primers, and one for TNF. Fromthis, three different standard curves were generated showing the numberof copies of total mtDNA (12s amplicon-total mitochondrial genomeprimers), 3.4 deletion or total nuclear DNA (TNF-single copy nucleargene primers). The C_(T) values of the samples were then converted tothe number of DNA copies by comparing the sample C_(T) to that of thestandards.

The determination of malignancy was based upon the quantity of the 3.4kb deletion present in the normalized sample as indicated by thelocation of the cycle threshold. This location may be either absolute,as in greater than 25 cycles but less than 30 cycles, or more likely aratio between the total mitochondrial DNA present as indicated by the12s amplicon, and the 3.4 kb deletion. This may be expressed as apercent of the total mitochondrial DNA.

In order to automate the analyses of these samples, bioinformatics toolswere employed. The three variables that were considered for theseanalyses were the cycle threshold C_(T) of Tumour Necrosis Factor (TNF),total species of mitochondria that contain those specific primer sites,and those mitochondria that harbour the deletion of interest.

Table 6 and FIG. 7 show the difference in the mean C_(T) scores forsamples from malignant tissue and benign tissue. The mean C_(T) valuefor normal tissue was 30.5889, while the mean C_(T) for malignant tissuewas 27.8533 thereby illustrating a difference in the quantity of mtDNAhaving the 3.4 kb deletion in malignant breast tissue compared to normalbreast tissue.

TABLE 6 Mean values for C_(T) scores Group Statistics Std. Std. GRP NMean Deviation Error Mean del3.4 normal 9 30.5889 2.53897 .84632malignant 9 27.8533 2.52253 .84084

FIG. 8 is an ROC curve illustrating the specificity and sensitivity ofthe 3.4 kb mtDNA deletion as a marker for breast cancer when testingbreast tissue. These results were obtained using a cutoff C_(T) of29.1900. The sensitivity of the marker at this C_(T) was 77.8%, whilethe specificity was 77.8%.

Table 7 shows the calculation of the area under the curve for thepresent example. As a measure of the accuracy of the test.

TABLE 7 Results Showing Area Under the Curve Area Under the Curve TestResult Variable(s): del3.4 Asymptotic 95% Std. Asymptotic ConfidenceInterval Area Error^(a) Sig.^(b) Lower Bound Upper Bound .790 .112 .038.570 1.010 ^(a)Under the nonparametric assumption ^(b)Null hypothesis:true area = 0.5

The determination of the cutoff C_(T) of 29.1900 is shown in table 8below. The results listed in table 8 show that a cutoff C_(T) of 29.1900provided the highest sensitivity and specificity at 78% and 78%respectively.

TABLE 8 Determination of C_(T) cutoff. Coordinates of the Curve TestResult Variable(s): del3.4 Positive if Less Than or Equal To ^(a)Sensitivity 1 - Specificity 24.6000 .000 .000 25.6800 .111 .000 25.7700.222 .000 25.9250 .333 .000 26.2050 .444 .000 26.8400 .556 .000 27.4800.556 .111 28.1600 .556 .222 28.8800 .667 .222 29.1900 .778 .222 29.4600.778 .333 29.8750 .778 .444 30.5850 .778 .556 31.2200 .778 .667 31.5000.889 .667 31.7650 .889 .778 32.9900 1.000 .778 34.3350 1.000 .88935.6400 1.000 1.000 ^(a) The smallest cutoff value is the minimumobserved test value minus 1, and the largest cutoff value is the maximumobserved test value plus 1. All the other cutoff values are the averagesof two consecutive ordered observed test values.

Example 5 The 3.4 Kb Deletion in the Prostate Massage Fluid ofIndividuals with Prostate Cancer as Compared to the Fluid from Thosewithout Histological Evidence of Prostate Cancer

Forty prostate massage fluid samples were collected by urologists frompatients who were either subsequently diagnosed with prostate cancer orshowed no histological evidence of prostate cancer following a prostateneedle biopsy procedure. The sample was deposited on a IsoCode Card™(Schleicher & Shuell), dried, and then extracted according to themanufacturer's protocol. All DNA extracts were quantified using aNanoDrop™ ND-1000 Spectrophotometer and the DNA concentration normalizedto 2 ng/ul. Each sample was then amplified according to the followingparameters:

1X iQ SYBR Green Supermix ™ (Bio-Rad P/N 170-8880)150 nmol forward primer (SEQ ID NO: 2)(5′-TAGACTACGTACATACTAACCCTACTCCTA-3′). 150 nmol reverse primer(SEQ ID NO: 3) (5′-GAGGTAGGATTGGTGCTGT-3′) 20 ng template DNAin a 25 ul reaction.

Reactions were cycled on an Opticon™ 2 DNA Engine (Bio-Rad Canada)according to the following protocol:

-   -   1. 95° C. for 3 minutes    -   2. 95° C. for 30 seconds    -   3. 66° C. for 30 seconds    -   4. 72° C. for 30 seconds    -   5. Plate Read    -   6. Repeat steps 2-5 44 times    -   7. 72° C. for 10 minutes    -   8. Melting Curve from 50° C. to 105° C., read every 1° C., hold        for 3 seconds    -   9. 10° C. Hold

TABLE 9 Results showing the mean C_(T) Values for Prostate Massage FluidTest Group Statistics Std. Std. Group N Mean Deviation Error Mean DEL3.4benign 25 37.1869 3.18495 .63699 malignant 15 33.7712 3.98056 1.02778

Tables 9 and 10 show a significant difference between the mean C_(T)values obtained for the benign sample and the malignant sample groups(p=0.005).

TABLE 10 Results Showing Difference (p = 0.005)for C_(T) values ofsamples. Independent Samples Test t-test for Equality of Means Levere'sTest for 95% Confidence Interval Equality of Variances Sig. Mean Std.Error of the Difference F Sig. t df (2-tailed) Difference DifferenceLower Upper DEL34 Equal variances 1.251 .270 2989 38 .005 341570 1.142831.10217 5.72923 assumed Equal variances 2825 24.696 .009 341570 1.20917.92382 5.90758 not assumed

FIG. 5 is a Receiver Operating Characteristic (ROC) curve illustratingthe specificity and sensitivity of the 3.4 kb mtDNA deletion as a markerfor prostate cancer when testing prostate massage fluid. These resultswere obtained using a cutoff C_(T) of 37.3683. The sensitivity of themarker at this C_(T) is 87%, while the specificity is 64%.

The accuracy of the test depends on how well the test separates thegroup being tested into those with and without the prostate cancer.Accuracy is measured by the area under the ROC curve. Table 11 shows thecalculation of the area under the curve for the present example.

TABLE 11 Results Showing Area Under the ROC Curve Area Under the CurveTest Result Variable(s): DEL3.4 Asymptotic 95% Std. AsymptoticConfidence Interval Area Error^(a) Sig.^(b) Lower Bound Upper Bound .768.074 .005 .622 .914 ^(a)Under the nonparametric assumption ^(b)Nullhypothesis: true area = 0.5

TABLE 12 Determination of Specificity and Sensitivity Coordinates of theCurve Test Result Variable(s): DEL3.4 Positive if Less Than or Equal To^(a) Sensitivity 1-Specificity 26.2992 .000 .000 27.3786 .067 .00028.2484 .133 .000 29.5193 .200 .000 30.1757 .200 .040 30.4580 .200 .08030.5980 .267 .080 31.5709 .333 .080 32.5712 .333 .120 32.9500 .333 .16033.3314 .400 .160 33.6547 .467 .160 33.9247 .533 .160 34.3554 .533 .20034.9056 .533 .240 35.4650 .533 .280 35.9172 .533 .320 36.0648 .600 .32036.3616 .667 .320 36.6421 .733 .320 36.8531 .733 .360 37.1188 .800 .36037.3683 .867 .360 37.5200 .867 .400 37.8341 .867 .440 38.2533 .867 .48038.5198 .933 .480 38.6519 .933 .520 38.8552 .933 .560 39.1258 .933 .60039.2734 .933 .640 39.4952 .933 .660 39.7323 1.000 .680 39.8956 1.000.720 41.0000 1.000 1.000 The smallest cutoff value is the minimumobserved test value −1, and the largest cutoff value is the maximumobserved test value plus 1. All the other cutoff values are the averageof two consecutive ordered, observed test values.

The determination of the cutoff C_(T) of 37.3683 is shown in table 12above. The results listed in table 12 illustrate that a cutoff C_(T) of37.3683 provided the highest sensitivity and specificity.

Example 6 The 3.4 Kb Deletion in the Urine of Individuals with ProstateCancer as Compared to the Fluid from Those without Histological Evidenceof Prostate Cancer

Urine samples were collected from 5 patients who were diagnosed withprostate cancer and 5 who have had a needle biopsy procedure which wasunable to detect prostate malignancy. These samples were collectedfollowing a digital rectal exam (DRE) to facilitate the collection ofprostate cells.

Upon receipt of the samples a 5 ml aliquot was removed and then 2 mlswere centrifuged at 14,000×g to form a pellet. The supernatant wasremoved and discarded. Pellets were resuspended in 200 ul phosphatebuffered saline solution. Both the resuspended pellet and the wholeurine sample were subjected to a DNA extraction procedure using theQiaAMP™ DNA Mini Kit (Qiagen P/N 51304) according to the manufacturer'sdirections. The resulting DNA extracts were then quantified using aNanoDrop™ ND-1000 Spectrophotometer and normalized to a concentration of0.1 ng/ul.

Samples were analyzed by quantitative real-time PCR with the 3.4 kbdeletion specific primers according to the following:

1X iQ SYBR Green Supermix ™ (Bio-Rad P/N 170-8880)100 nmol forward primer (SEQ ID NO: 2)(5′-TAGACTACGTACATACTAACCCTACTCCTA-3′) 100 nmol reverse primer(SEQ ID NO: 3) (5′-GAGGTAGGATTGGTGCTGT-3′) 1 ng template DNAin a 25 ul reaction.

Reactions were cycled on an Opticon™ 2 DNA Engine (Bio-Rad Canada)according to the following protocol:

1. 95° C. for 3 minutes

2. 95° C. for 30 seconds

3. 69° C. for 30 seconds

4. 72° C. for 30 seconds

5. Plate Read

6. Repeat steps 2-5 44 times

7. 72° C. for 10 minutes

8. Melting Curve from 50° C. to 105° C., read every 1° C., hold for 3seconds

9. 10° C. Hold

TABLE 13 Mean values for C_(T) scores Group Statistics Std. Std.GRPfluid38 N Mean Deviation Error Mean CTf Benign 5 33.2780 1.10900.49596 Malignant 5 30.6980 2.55767 1.14382

Tables 13 and 14 show a significant difference between the mean C_(T)values obtained for benign sample and the malignant sample groups(p=0.005).

TABLE 14 Results Showing Difference (p = 0.005) for C_(T) values ofsamples. Independent Samples Test t-test for Equality of Means Levere'sTest for 95% Confidence Interval Equality of Variances Sig. Mean Std.Error of the Difference F Sig. t df (2-tailed) Difference DifferenceLower Upper CTf Equal variances 1.272 .292 2069 8 .072 258000 124672−.29494 5.45494 assumed Equal variances 2069 5453 .089 258000 124672−.54639 5.70639 not assumed

FIG. 6 is a Receiver Operating Characteristic (ROC) curve illustratingthe specificity and sensitivity of the 3.4 kb mtDNA deletion as a markerfor prostate cancer when testing urine. These results were obtainedusing a cutoff C_(T) of 31.575. The sensitivity of the marker at thisC_(T) is 80%, while the specificity is 100%.

The determination of the cutoff C_(T) of 31.575 is shown in table 15.The results listed in table 15 show that a cutoff C_(T) of 31.575provided the highest sensitivity and specificity.

TABLE 15 Determination of C_(T) cutoff. Coordinates of the Curve TestResult Variable(s): CTf Positive if Less Than or Equal To ^(a)Sensitivity 1 - Specificity 26.2900 .000 .000 28.4950 .200 .000 30.3850.400 .000 31.0800 .600 .000 31.5750 .800 .000 32.1400 .800 .200 32.8150.800 .400 33.8700 .800 .600 34.3350 .800 .800 34.3550 1.000 .800 35.37001.000 1.000 ^(a) The smallest cutoff value is the minimum observed testvalue minus 1, and the largest cutoff value is the maximum observed testvalue plus 1. All the other cutoff values are the averages of twoconsecutive ordered observed test values.

Example 7 Detection of Re-Circularized 3.4 Kb Deleted Sequence inProstate Malignant and Benign Tissue

In this example, the amount of re-circularized 3.4 kb deleted mtDNAmolecules in samples was tested as an indicator for prostate cancer. Asmentioned above, the 3.4 kb sequence, upon deletion, may reform as acircular mtDNA molecule. Amplification of a target region from thedeleted 3.4 kb mtDNA sublimon was conducted using a primer pair (SEQ IDNOS: 9 and 10). The forward primer (SEQ ID NO: 9), overlaps therejoining site of the ends of the 3.4 kb sequence.

Prostate tissue was formalin-fixed paraffin embedded prostate tissueneedle biopsies.

The reagent setup used for this example was as follows:

250 nmol each primer

12.5 ul of 2λ reaction mix,

20 ng (10 ul of 2 ng/ul) template in 25 ul reaction volume.

The cycling parameters were as follows:

1. 95 degrees Celsius for 3 minutes

2. 95 degrees Celsius for 30 seconds

3. 62 degrees Celsius for 30 seconds

4. 72 degrees Celsius for 30 seconds

5. Plate Read

6. Repeat steps 2-5 44 times

7. 72 degrees for 10 minutes

8. Melting Curve from 50-100 degrees, reading every 1 degree for 3seconds

9 4 degrees HOLD.

Amplification of a target region from the deleted 3.4 kb mtDNA sublimonwas conducted using a primer pair (SEQ ID NOS: 9 and 10).

Table 16 below provides a summary of testing conducted for the detectionof the actual 3.4 kb deleted in mtDNA obtained from malignant and benignprostate tissue. Using a C_(T) score of 30.0, a clear identification ofmalignant and benign tissue was possible. As such, an increase in theamount of the 3.4 kb molecule present in a sample was indicative ofcancer.

TABLE 16 C_(T) scores for Detection of Cancer in Prostate TissueDescription C_(T) Benign sample 1 33.75 Malignant sample 1 28.79 Benignsample 2 30.96 Malignant sample 2 28.4 Benign sample 3 32.19 Malignantsample 3 27.38

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention as outlined in the claims appended hereto.

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What we claim is:
 1. A method of detecting a cancer in an individualcomprising; a) obtaining a biological sample from the individual; b)quantifying the amount of mtDNA in the sample having a deletion in thenucleic acid sequence spanning approximately residues 10744 and 14124 ofthe mtDNA genome; c) comparing the amount of mtDNA in the sample havingthe deletion to the amount of the deletion in a reference sample ofmtDNA wherein the reference sample is from a known non-cancerous tissueor body fluid or from a known cancerous tissue or body fluid; wherein ifthe reference sample is from a known non-cancerous tissue or body fluid,then an elevated level of the deletion in the biological sample comparedto the non-cancerous reference sample is indicative of cancer and if thereference sample is from a known cancerous tissue or body fluid samplethen an equivalent or elevated level of the deletion in the biologicalsample compared to the cancerous reference sample is indicative ofcancer.
 2. The method of claim 1 wherein the deletion has a nucleic acidsequence corresponding to the sequence identified in SEQ ID NO:
 1. 3.The method of claim 1 wherein the quantifying of the deletion includesamplifying a target region of mtDNA that is indicative of the deletion,and quantifying the amount of the amplified target region.
 4. The methodof claim 3 wherein a primer having a sequence corresponding to SEQ IDNO: 2 is used as part of a pair of amplification primers for amplifyingthe target region.
 5. The method of claim 1 wherein the step ofquantifying is conducted using real-time PCR.
 6. The method of claim 1wherein the cancer is prostate cancer.
 7. The method of claim 1 whereinthe cancer is breast cancer.
 8. The method of claim 1 wherein thebiological sample is a body tissue or body fluid.
 9. The method of claim8 wherein the biological sample is breast tissue, prostate tissue,prostate massage fluid, or urine.
 10. The method according to claim 3wherein the amplifying of the target region is conducted using a pair ofamplification primers, one of the pair of amplification primersoverlapping a splice joining regions on opposite ends of the deletion.11. A method of monitoring an individual for the development of a cancercomprising; a) obtaining a biological sample; b) quantifying the amountof mtDNA in the sample having a deletion in the nucleic acid sequencespanning approximately residues 10744 and 14124 of the mtDNA genome; c)repeating steps a) to b) over a duration of time; and d) wherein anincreasing level of the deletion over the duration of time is indicativeof cancer.
 12. The method of claim 11 wherein the deletion has a nucleicacid sequence corresponding to the sequence identified in SEQ ID NO: 1.13. The method of claim 11 further comprising at least one step selectedfrom the group consisting of: (a) comparing the amount of mtDNA in thesample having the deletion to the amount of the deletion in a referencesample of mtDNA from known non-cancerous tissue or body fluid; and (b)comparing the amount of mtDNA in the sample having the deletion to theamount of the deletion in a reference sample of mtDNA from knowncancerous tissue or body fluid.
 14. The method of claim 11 wherein thequantifying of the deletion includes amplifying a target region of mtDNAthat is indicative of the deletion, and quantifying the amount of theamplified target region.
 15. The method of claim 11 wherein the step ofquantifying is conducted using real-time PCR.
 16. The method of claim 14wherein a primer having a sequence corresponding to SEQ ID NO: 2 is usedas part of a pair of amplification primers for amplifying the targetregion.
 17. The method of claim 11 wherein the cancer is prostatecancer.
 18. The method of claim 11 wherein the cancer is breast cancer.19. The method of claim 11 wherein the biological sample is a bodytissue or body fluid.
 20. The method of claim 19 wherein the biologicalsample is breast tissue, prostate tissue, prostate massage fluid, orurine.
 21. A method of detecting a cancer in an individual comprising;a) obtaining a biological sample from the individual; b) quantifying theamount of mtDNA in the sample having a sequence corresponding to thesequence identified in SEQ ID NO: 1; c) comparing the amount of mtDNA inthe sample corresponding to SEQ ID NO: 1 to at least one known referencevalue, wherein the at least one known reference value is the amount ofmtDNA corresponding to SEQ ID NO: 1 in a reference sample of mtDNA fromknown non-cancerous tissue or body fluid or a reference sample of mtDNAfrom known cancerous tissue or body fluid, and wherein if the referencevalue is from a reference sample of mtDNA from known non-canceroustissue or body fluid then an elevated amount of the mtDNA correspondingto SEQ ID NO: 1 in the biological sample compared to the non-cancerousreference sample is indicative of cancer and wherein if the referencevalue is from a reference sample of mtDNA from known cancerous tissue orbody fluid then an equivalent or elevated amount of the mtDNAcorresponding to SEQ ID NO: 1 in the biological sample compared to thecancerous reference sample is indicative of cancer.
 22. The method ofclaim 21 wherein the step of quantifying is conducted using real-timePCR.
 23. The method of claim 21 wherein the quantifying of the deletionincludes first amplifying a target region of mtDNA that is indicative ofthe deletion, and quantifying the amount of the amplified target region.24. The method of claim 21 wherein one of a pair of primers used in theamplifying of the target region overlaps a rejoining site of thesequence corresponding to SEQ ID NO: 1, after the sequence hasre-circularized.
 25. The method of claim 23 wherein a primer having asequence corresponding to SEQ ID NO: 9 is used as part of a pair ofamplification primers for amplifying the target region.
 26. The methodof claim 21 wherein the cancer is prostate cancer.
 27. The method ofclaim 21 wherein the cancer is breast cancer.
 28. The method of claim 21wherein the biological sample is a body tissue or body fluid.
 29. Themethod of claim 28 wherein the biological sample breast tissue, prostatetissue, prostate massage fluid, or urine.
 30. The method of claim 22wherein the reference value is a cycle threshold.
 31. A method ofdetecting a deletion spanning approximately nucleotides 10744 to 14124of the human mtDNA genome, wherein said deletion is associated withprostate cancer, in a subject having mtDNA, comprising: a) providing abiological sample from the subject; b) detecting the presence of thedeletion in the mtDNA, wherein presence of the deletion in the mtDNAindicates prostate cancer, a predisposition to prostate cancer or theprogression of prostate cancer.
 32. The method of claim 31, wherein thestep of detecting the presence of the deletion in the mtDNA comprisesPCR analysis using a primer that bridges the mtDNA junction formed bythe deletion spanning approximately nucleotides 10744 to
 14124. 33. Amethod of detecting a mtDNA deletion spanning approximately nucleotides10744 to 14124 of the human mtDNA genome as a biomarker for prostatecancer, the method comprising: a) providing a biological sample from thesubject; b) detecting the presence of the deletion in the mtDNA; c)comparing the amount of mtDNA in the sample having the deletion to theamount of the deletion in a reference sample of mtDNA wherein thereference sample is from a known non-cancerous tissue or body fluid orfrom a known cancerous tissue or body fluid; wherein if the referencesample is from a known non-cancerous tissue or body fluid, then anelevated level of the deletion in the biological sample compared to thenon-cancerous reference sample is a biomarker indicative of cancer andif the reference sample is from a known cancerous tissue or body fluidsample then an equivalent or elevated level of the deletion in thebiological sample compared to the cancerous reference sample is abiomarker indicative of cancer.
 34. The method of claim 33, wherein thedeletion is approximately 3379 bp.
 35. A method for confirming orrefuting a prostate cancer biopsy test from a biopsy sample, comprising:a) obtaining a normal tissue from a biopsy sample; b) detecting theabsence or presence of a human mtDNA deletion at approximatelynucleotides 10744 to 14124 of the human mtDNA genome in the normaltissue; and c) comparing the amount of the mtDNA deletion in the normaltissue to the amount of the deletion in a reference sample of mtDNAwherein the reference sample is from a known non-cancerous tissue orbody fluid or from a known cancerous tissue or body fluid; wherein ifthe reference sample is from a known non-cancerous tissue or body fluid,then an elevated level of the deletion in the normal tissue compared tothe non-cancerous reference sample is indicative of a cancerous biopsyand if the reference sample is from a known cancerous tissue or bodyfluid sample then an equivalent or elevated level of the deletion in thebiological sample compared to the cancerous reference sample isindicative of a cancerous biopsy.